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A mammalian family of lipid hydrolases, designated “patatin-like phospholipase domain containing (PNPLA)” recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

NTE-related esterase (NRE), conserved in mouse, rat and human, was a member of patatin-like phospholipases (PLPLA) with high homology to neuropathy target esterase (NTE). Little has been known about the characteristics of NRE and NRE functional esterase activity has yet not been defined. The C-terminal gene sequence of mouse NRE (mNREC) encoding 923-1,326 amino acid containing the patatin domain was first cloned and then expressed tagged with enhanced green fluorescence protein (EGFP) in mammalian cells. The results showed that mNREC had NTE esterase activity in mammalian cells. Overexpression of mNREC did not affect the esterase activity sensitive to paraoxon or resistant to both paraoxon and mipafox. mNREC was distributed in the cytoplasm in contrast to the distribution of human NTE esterase domain. The expression analysis of NRE gene in adult mouse tissues by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that there were higher levels of NRE mRNA in the brain and testis than in the liver and kidney, which was about 50% and 35% of that in the brain. These results firstly showed the tissue distribution of NRE gene in adult mouse and defined that NRE had functional esterase activity.

Neuropathy target esterase (NTE) was originally identified as the primary target site of those organophosphorus compounds that induce delayed neuropathy in human and some animals. Here we examined the role of protein kinase C (PKC) in the regulation of the NTE activity in mammalian cells. Six-hour exposure of human neuroblastoma SK-N-SH cell to a PKC activator phorbol 12-myristate 13-acetate (PMA) decreased the activity of NTE, and this effect was blocked by the PKC inhibitor staurosporine. These results suggest that PKC down-regulates the activity of NTE. NTE protein levels were down-regulated by PMA-stimulation as detected by Western blot analysis using the NTE-specific antibody, which resulted from down-regulation of NTE mRNA level as verified by real-time reverse transcription polymerase chain reaction (RT-PCR). However, there were no changes in the activity or protein levels of stable expression of NTE esterase activity domain (NEST) in SK-N-SH cells and transient expression of full-length NTE construct in COS7 cells driven by cytomegalovirus (CMV) promoter rather than by the cell's own one, despite the absence or presence of PMA stimulation. Together, these findings suggest that stimulation with PMA reduces the expression of NTE mRNA levels but does not affect the exogenous promoter-driven NTE expression in mammalian cells.

Cytoskeletal components play an important role in maintaining cellular architecture and internal organization, with clear involvement of defining cell shape, in cell division and other cellular processes, such as neurite extension and maintenance. Alterations of cytoskeleton in human neuroblastoma SK-N-SH cells after exposure to different concentrations of tri-ocresyl phosphate (TOCP) for 12 hr were investigated. TOCP decreased the cell viability in a dose-dependent manner; the viability of SK-N-SH was reduced to approximately 50% of baseline after a 12-hour exposure to TOCP at high concentration (5 mM). Biochemical characterization by western blotting revealed that 1 and 5 mM concentrations of TOCP significantly inhibited the expression of neurofilament high molecular weight protein (NF-H), and that 5 mM TOCP inhibited expression of microtubule-associated protein 2c and tau protein, but not beta-actin. Indirect immunofluorescence analysis revealed that higher concentrations of TOCP decreased the length of neuritis and changed the structure of microfilaments, which are associated with NF-H. In addition, activities of neuropathy target esterase and acetylcholinesterase were significantly reduced after exposure to 5 mM TOCP for 12 hr. Together, these results suggested that the loss of cytoskeletal components is the early event during the process of TOCP toxicity towards human neuroblastoma SK-N-SH cells.

Neuropathy target esterase (NTE) is phosphorylated and aged by oraganophosphorus compounds (OP) that induce delayed neuropathy in human and some animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neural differentiation. However, to date, there is no direct evidence of the relevance of NTE in neural differentiation under physiological conditions. In this study we have investigated a possible role for NTE in the all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells by antisense RNA. A NTE antisense RNA construct was generated and then transfected into human neuroblastoma SK-N-SH cells. A positive cell clone that can stably express NTE antisense RNA was obtained by G418 selection and then identified by western blotting. NTE activity was depressed in the transfected cells with only about 50% activity of the enzyme in the control cells. ATRA-induced differentiation of the neuroblastoma cells with lowered NTE activity revealed that inhibition of NTE expression does not affect neural differentiation in SK-N-SH cells. The result suggested that organophosphates may inhibit neural differentiation by initially acting on other targets other than NTE.